The present invention relates to compounds of the oligonucleotide type, as well as to their applications.
Antisense oligonucleotides are short synthetic DNA or RNA molecules of sequence complementary to a target sequence belonging to a gene or to a messenger RNA whose expression it is desired to block specifically. Antisense oligonucleotides may be directed towards a messenger RNA sequence, or alternatively towards a DNA sequence. Antisense oligonucleotides hybridize with the sequence to which they are complementary and can thus block the expression of the messenger RNA carrying this sequence.
The term xe2x80x9coligonucleotidexe2x80x9d is used in a general manner to denote a polynucleotide of ribo- or deoxyribo-series. Where the issue of a particular property linked to the use of a deoxyribo-series or a ribo-series is involved, the complete name oligodeoxyribonucleotide or oligoribonucleotide may be used. An oligonucleotide can be single-stranded, that is to say contain only one line of nucleotides which are not paired with another chain, or can alternatively be double-stranded, that is to say contain nucleotides paired with another polynucleotide chain. Two complementary oligonucleotides form a double-stranded structure. A single-stranded oligonucleotide can, however, possess double-stranded regions by intra-chain pairings between complementary sequences carried on the same strand.
The term hybridization used here means the formation of hydrogen bonds between pairs of complementary bases, guanine and cytosine forming three hydrogen bonds and adenine and thymine forming two.
Antisense oligonucleotides are synthesized chemically, and frequently contain modifications which change the actual skeleton of the molecule or carry additional reactive groups localized at their ends. The objectives of these modifications introduced into anti-sense oligonucleotides are either to enhance the resistance of these molecules to nucleolytic degradation, or to promote their interactions with their targets, or to permit specific degradation/modification reactions of the RNA or DNA targets, or to increase their intracellular penetration.
Antisense oligonucleotides are sensitive to nuclease degradation, and mainly to the action of exonucleases. Nucleases occur in all compartmentsxe2x80x94cellular and extracellular, especially in the serumxe2x80x94and cause a rapid degradation of these molecules. A pharmacological use of antisense molecules involves solving these problems of degradation in order to achieve satisfactory pharmacokinetics and hence an adequate perpetuation of the effects of these molecules. Many chemical modifications enable antisense oligonucleotides to become nuclease-resistant. Some modifications directly affect the structure or nature of the phosphodiester bond (methylphosphonates, phosphorothioates, alpha-oligonucleotides, phosphoramidates, to mention a few examples), other [sic] consist in adding blocking groups to the 3xe2x80x2 and 5xe2x80x2 ends of the molecules (Perbost et al., 1989; Bertrand et al., 1989; Bazile et al., 1989; Andrus et al., 1989; Cazenave et al., 1989; Zon, 1988; Maher and Dolnick, 1988; Gagnor et al., 1987; Markus-Sekura, 1987).
To increase the efficacy of the interactions between an oligonucleotide and its target, an inter-calating group (acridines for example) may be added to one end of the antisense oligonucleotide. Lastly, re-active groups (alkylating agents, psoralens, Fe-EDTA for example) capable of causing cleavages or permanent chemical changes in the target may be added to the antisense oligonucleotides (Sun et al., 1989; Helene, 1989; Durand et al., 1989; Sun et al., 1988; Helene and Thuong, 1988; Verspieren et al., 1987; Sun et al., 1987; Cazenave et al., 1988, 1987; Le Doan et al., 1987; Toulme et al., 1986; Vlassov et al., 1986).
The last type of conventional modification of antisense oligonucleotides consists in adding groups which modify the charge and/or hydrophilicity of the molecules in order to facilitate their passage through the membrane (Kabanov et al., 1990; Degols et al., 1989; Stevenson et al., 1989; Leonetti et al., 1988).
All these modifications can obviously be combined with one another.
Not all the regions of a messenger RNA are sensitive in a like manner to the effects of an antisense oligonucleotide. A messenger RNA is not a set linear molecule but, on the contrary, a molecule possessing many secondary structural features (complex intramolecular hybridizations) and tertiary structural features (refoldings and particular conformations, pseudo-nodes), and which interacts with structural and functional nucleo-proteins (basic proteins, splicing, polyadenylation and capping complexes, translation complex for example). The effective availability and accessibility of the different regions of a messenger RNA will depend on their engagement in these structural features. Correspondingly, the efficacy of an inhibitory agent which interacts with this or that sequence will also depend on the engagement of this sequences [sic] in a particular function. The target regions for antisense molecules must be accessible to the oligonucleotide.
The use of software for prediction of secondary structures enables theoretical degrees of accessibility to be predicted, and hence the choice of targets for antisense oligonucleotides to be guided. As a whole, the regions most widely used as targets are translation initiation sites (AUG initiation region) and also splicing sites (SD/SA junctions). Many other sequences not having particular functional properties and not engaged in intramolecular pairing have also proved effective as a target for antisense oligonucleotides (see the examples mentioned later).
Antisense oligodeoxyribonucleotides may also be directed towards certain regions of double-stranded DNA (homopurine/homopyrimidine sequences or purine/pyrimidine-rich sequences), and can thus form triple helices (Perroualt et al., 1990; Franxc3xa7ois et al.(A), 1989; Franxc3xa7ois et al.(B), 1989; Franxc3xa7ois et al.(C), 1989; Wang et al., 1989; Maher et al., 1989; Sun et al., 1989; Boidot-Forget et al., 1988; Moser and Dervan, 1987; Dervan, 1986). Oligonucleotides directed in this manner towards DNA have been termed xe2x80x9canti-genexe2x80x9d or alternatively xe2x80x9canti-codexe2x80x9d. The formation of a triple helix at a particular sequence can block the binding of proteins involved in the expression of a gene and/or permit the introduction of irreversible damage into the DNA if the oligonucleotide in question possesses a particular reactive group. Such antisense oligonucleotides can become true artificial restriction endonucleases, directed on request towards specific sequences.
The hybridization between an antisense oligonucleotide and a target messenger RNA can block expression of the latter in several ways, either sterically or pseudocatalytically (Gagnor et al., 1989; Jesus et al., 1988; Markus-Sekura, 1987):
the interaction between the messenger RNA and a complementary antisense oligonucleotide can create a physical barrier preventing the binding and/or progression of proteins or protein complexes needed for translation, maturation, stabilization or transport of the messenger RNA. This physical blockade will lead finally to an inhibition of the expression of the target messenger RNA.
the hybridization between a messenger RNA and an antisense oligodeoxyribonucleotide will create a substrate for RNase H, an enzyme present in all eukaryotic cells. RNase H is an enzyme which specifically degrades RNA when it is hybridized with DNA. The hybridization of an antisense oligonucleotide with a target RNA will hence lead to cleavage of this target RNA at the location of this hybridization, and hence to its permanent inactivation.
moreover, as stated above, antisense oligonucleotides can contain reactive groups capable of directly producing irreversible damage in the target RNA molecule.
As regards antisense oligonucleotides directed towards DNA, these can act either by inhibiting the binding of a regulatory protein essential to expression of the target gene (transcription factor for example), or by producing irreversible damage (cleavages, cross-links) in the DNA molecule, making it locally incapable of genetic expression.
Ribozymes are RNA molecules endowed with enzymatic activity, capable, in particular, of causing endonuclease cleavages in target RNAs. A ribozyme may be considered to be a particular antisense oligonucleotide endowed with a natural endonuclease catalytic activity (Vasseur, 1990; Symons, 1989; Jeffrie and Symons, 1989; Haseloff and Gerlach, 1988; Uhlenbeck, 1987; Symons et al., 1987). Typically, a ribozyme consists of two portions; on the one hand it contains a sequence complementary to the target sequence which it is desired to cut, and on the other hand a catalytic sequence functioning as a reactive group (Fedor and Uhlenbeck, 1990; Uhlenbeck et al., 1989; Sheldon and Symons, 1989; Sampson et al., 1987). It is possible at the present time, using a consensus active site deduced from the sequence of viral ribozymes, to cut theoretically any messenger RNA at a predetermined position (Haseloff and Gerlach, 1988; Uhlenbeck, 1987). Ribozymes encounter the same problems of use as conventional antisense oligonucleotides, especially as regards the phenomena of degradation, RNAs being still more sensitive to nucleolytic degradation than DNAS.
Antisense oligonucleotides make it possible to block specifically the expression of cell messenger RNAs, for example oncogenic type messengers (Tortora et al., 1990; Chang et al., 1989; Anfossi et al., 1989; Zheng et al., 1989; Shuttleworth et al., 1988; Cope and Wille, 1989; Cazenave et al., 1989) and many different types of viral messenger RNAs originating from viruses as varied as VSV (Degols et al., 1989; Leonetti et al., 1989), SV40 (Westermann, et al., 1989), influenza viruses (Kabanov et al., 1990; Zerial et al., 1987), the encephalomyocarditis virus (Sankar et al., 1989), adenovirus (Miroschnichenko et al., 1989), HSV (Gao et al., 1988) and HIV (Matzukura et al., 1989; Stevenson et al., 1989; Matzukura et al., 1988; Goodchild et al., 1988).
With ribozymes, it is possible to cleave in vivo the messenger RNA coding for the CAT marker gene (Cameron and Jennings, 1989), to inhibit the process of maturation of histone messenger RNA (Cotten et al. 1989; Cotten and Birnstiel, 1989) or to protect the cell partially against HIV-1 infection (Sarver et al., 1990).
Oligonucleotides may also be used in the context of xe2x80x9csensexe2x80x9d type strategies. This approach consists in using a single-stranded or double-stranded oligonucleotide of the deoxyribo- or ribo-series, of specific sequence, as an agent for binding a protein possessing an affinity for this sequence and whose effective concentration inside the cells it is desired to decrease, by competition. It is thus possible to envisage using oligonucleotides which interact with transcription factors, viral encapsidation factors, translation regulation factors, and the like. This approach is not yet exploited, unlike the more conventional antisense strategy. In this case, the problem of the stability of the oligonucleotides is also a critical important factor for the efficacy and durability of their action. The use of modified oligonucleotides for such an approach can run into structural problems of recognition by the proteins. The possibility of having at one""s disposal natural oligonucleotides which are stable in serum and cells would make it possible to envisage the development of novel therapeutic methods targeted especially on the regulation factors having affinity for nucleic acids.
Antisense, and sense, oligonucleotides are hence potent and highly specific potential pharmacological agents which make it possible to inhibit the expression messengers coding for products exerting pathogenic effects.
The therapeutic use of oligonucleotides runs, however, into several problems of a physiological type, especially that of the intracellular delivery of these molecules and that of their sensitivity to nucleolytic degradation. The use of modified derivatives enables the problem of nuclease sensitivity to be overcome, but introduces a further problem, that of the possible toxicity of the chemical modifications introduced into the molecule.
The use of modified antisense oligonucleotides creates, in effect, problems of a toxicological nature. While some of the modifications are said to be fairly neutral, most are not without potential toxicity.
Chemically modified antisense oligonucleotides can possess a toxicity at several level [sic], either directly through effects of the whole molecule, or indirectly via the effects of the degradation products. Nucleotides carrying chemical modifications and which are present in a cell at a high concentration can thus possess a toxicityxe2x80x94and more especially a genotoxicityxe2x80x94which is not insignificant from a pharmacological standpoint.
For example, many problems raised by the use of modified antisense oligonucleotides, especially non-sequence-specific antiviral effects, appear indeed to be due to the nature of some of the chemical modifications introduced into the antisense oligonucleotides to make them nuclease-resistant.
From a toxicological standpoint, it is hence obvious that, the less the natural structure of the oligonucleotide is modified, the lower the risk of being confronted by pharmacological problems. A natural DNA or RNA molecule, as well as its degradation products, creates little or no problem of toxicology and of pharmacokinetics, which is not the case with a modified structure capable of giving, after being metabolized, multifarious potentially toxic derivatives.
It would hence be advantageous to be able to have at one""s disposal natural oligonucleotides containing only normal deoxy- or ribonucleotides linked to one another via a normal phosphodiester bond but possessing, however, a resistance to degradation.
The subject of the invention presented here is a novel structural type of antisense, or sense, oligonucleotide which is resistant to exonucleases without the involvement of stabilizing chemical modifications. The oligonucleotides forming the subject of the invention have the feature of possessing a closed structure which does not offer an end available to exonuclease degradation.
Such oligonucleotides may be used in their natural state but can, however, also contain modified nucleotides or reactive groups, or be physically combined with other molecules or macromolecules with the object of fortifying their efficacy of inhibition, their penetration, their affinity for their targets or their cellular or intracellular targeting, or for optimizing any other property.
In cells and, still more, in the body, in the blood circulation for example, natural antisense oligonucleotides are sensitive to nucleases. Nucleases are degradative enzymes capable of cutting the phosphodiester bonds of DNA or RNA, either by introducing internal cleavages in single- or double-stranded molecules, or by attacking these molecules from their ends. Enzymes which attack internally are termed endonucleases, and those which attack by the ends are termed exonucleases.
The stability of antisense oligonucleotidesxe2x80x94and hence their efficacyxe2x80x94may be considerably enhanced by introducing various chemical modifications making them resistant to degradation, as described above.
It is established that exonucleases are the species which are the main cause of degradation of antisense oligonucleotides in serum and in the cell. More especially, it appears that exonucleases attacking at the 3xe2x80x2-OH end are implicated most particularly in this phenomenon.
Modifications made to the structure of the ends of antisense oligonucleotides can protect them, block exonuclease activity and confer an increased stabilization on the oligonucleotides.
The invention described here is based on the novel idea that closed oligonucleotides not possessing free ends will hence be, by definition, resistant to this type of degradation. Closed, for example circular, oligonucleotides do not afford a substrate which is accessible to 3xe2x80x2 or 5xe2x80x2 exonucleases, and are hence thereby stabilized.
More especially, the invention hence relates to an antisense or sense agent of the oligonucleotide type, which consists of one or more single-stranded oligonucleotide sequence(s) whose ends are linked to one another via covalent links to form at least partially a closed, single-stranded structure.
These agents are sometimes designated hereinafter closed oligonucleotides or circular oligonucleotides, inasmuch as this type of compound preferably possesses a majority of nucleotides relative to non-nucleotide structures.
The definition of the term oligonucleotide has been given above, and incorporates both the natural ribo- and deoxyribo-series, as well as the modifications of these bases which will be designated as a whole hereinafter unnatural and which have also been mentioned above.
The covalent link can be a non-nucleotide covalent structure of the protein, lipid or glycoside type and/or a mixed structure, as will be explained below. It is nevertheless preferable to use a nucleotide covalent structure, that is to say a phosphodiester bond.
The invention relates to closed, for example circular, oligonucleotides not having free ends, but composed of a succession of nucleotide bases bonded to one another via all types of bonds, and preferably via phosphodiester type bonds. These bases may be combined with one another via bonds such that the distance between these bases will be approximately 3 xc3x85 to 4 xc3x85, which is the distance found in natural DNA or RNA molecules when the internucleotide bonds are provided by phosphodiester groups. The closed, for example circular, oligonucleotides will advantageously be composed of natural nucleotides preferably bonded to one another via unmodified phosphodiester bonds, but may also contain modified nucleotides and/or modified bonds which comply with the distances between bases and permit the helical axial rotations characteristic of nucleic acid conformations. The closed oligonucleotides will hence be advantageously composed of the bases A, T, G, C, or U, in their deoxy- or ribonucleotide forms. The closed oligonucleotides may hence be either oligodeoxyribonucleotides or oligoribo nucleotides or mixed molecules containing deoxyribonucleotides and ribonucleotides.
The invention hence comprises any closed, single-stranded DNA or RNAxe2x80x94or mixed DNA/RNAxe2x80x94molecule, circular or possessing a circular portion, obtained biologically, chemically or by methods combining the techniques of synthetic chemistry with those of biology and biochemistry, possessing a resistance to exonucleases greater than that of an oligonucleotide of the same sequence but completely linear, not possessing a closed structure.
Examples of closed oligonucleotides are shown in FIGS. 1A to 1C.
FIG. 1A shows examples of closed antisense oligonucleotide structure. FIG. 1B shows examples of closed sense oligonucleotide structure. FIG. 1C shows a mixed molecule capable of exerting a sense and antisense effect.
The number of single nucleotides making up the closed oligonucleotides can vary widely, in particular between 10 and 200, but, as a guide, this number will advantageously be between about twenty and about fifty nucleotides, depending on the closing structure (circular, lasso or balloon structure, structure made double-stranded, and the likexe2x80x94see the descriptions of the different structures later), the uses (anti-RNA antisense molecule, anti-DNA antisense molecule, anti-protein sense molecule), the type of oligonucleotidexe2x80x94deoxy- or ribonucleotidexe2x80x94(simple antisense or ribozyme antisense, and the like) and the targets in question (messenger RNA, premessenger RNA, particular secondary structure, and the like).
The closed oligonucleotides forming the subject of the present invention are preferably composed of a sequence of bases containing adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), having the general formulae shown in FIG. 1D.
The closed oligonucleotides may also contain rare nucleotides (inosine, I, or rI for example) or modified nucleotides, either of the deoxyribo- or the ribo-series.
The closed oligonucleotides may contain nucleotides modified at the phosphodiester bond, for example according to the various formulae shown in FIG. 2. For example, the closed oligonucleotides may contain one or more of the well known groups which are phosphorothioates, methylphosphonates, phosphorodithioates, phosphoselenates, phosphoramidates and alkyl phosphotriesters. It should be noted, however, that the closed antisense oligonucleotides will preferably comprise natural nucleotides linked to one another via unmodified phosphodiester bonds.
The closed oligonucleotides may contain reactive nucleotides capable of establishing links with the sequence of the target molecule complementary to the oligonucleotide.
Thus, the closed oligonucleotides may carry reactive groups grafted onto the nucleotides, such as, for example, psoralen groups, or other bridging agents or intercalating agents capable of reacting with the sequence of the target molecule complementary to the oligonucleotide (see the various non-exhaustive examples shown in FIG. 2).
The closed antisense oligonucleotides may also include, among the nucleotides forming part of the closed structure, an RNA sequence possessing a catalytic activity. These circular oligonucleotides will thus be circular ribozymes, with joined ends, containing, in addition to the catalytic sequence which can be any RNA sequence capable of causing a cleavage or a modification in a target RNA sequence, an antisense type sequence complementary to the target sequence and which can consist either of RNA or of DNA or of a DNA/RNA mixture (FIG. 3).
Closed oligonucleotides coupled to molecules enabling their intracellular penetration to be increased, and especially lipophilic groups, polypeptides or proteins, also form part of the invention.
The closed oligonucleotides possess the main feature of not offering a substrate to 3xe2x80x2 and 5xe2x80x2 exonucleases. To acquire this property, the simplest structure is a circular oligonucleotide formed from a succession of nucleotides linked to one another via phosphodiester bonds, as indicated diagrammatically above, and possessing or otherwise intra-chain pairings (FIGS. 1A to 1C and FIG. 4).
Other structures of molecules closed with a non-3xe2x80x2-51 phosphodiester bond may also be synthesized and can possess a partial or total resistance to exonucleases.
Lasso-shaped molecules composed of deoxy- or ribonucleotide residues and closed either with a bond involving either the 3xe2x80x2 end or the 5xe2x80x2 end of the molecule (FIGS. 1A to 1C and FIG. 4B) also form part of the invention. In these lasso-shaped molecules, the linear portion can contain only a single nucleotide residue or alternatively can contain a nucleotide side chain of several residues. In these structures, one of the terminal nucleotides of the oligonucleotide is coupled to an internal nucleotide via a bond which can be formed with the base or with the sugar or with the phosphodiester group. Such oligonucleotides possess a free end and a blocked end. Resistance to exonucleases will hence be partial, it being possible for the free end to be subject to a nucleolytic degradation. The 5xe2x80x2 and 3xe2x80x2 ends of oligonucleotides are not sensitive in an equivalent manner to degradation, the 3xe2x80x2 end being more sensitive. A lasso-shaped circular oligonucleotide possessing only the 5xe2x80x2 end free is largely protected against degradation. In addition, exonucleases capable of degrading the linear portion of a lasso will be stopped at the branching point. At this stage, the oligonucleotide has become completely resistant to 3xe2x80x2 and 5xe2x80x2 exonucleases, irrespective of which end was initially free or branched. Such lasso-shaped molecules can contain natural or modified nucleotide residues, as stated above in the previous section. The general structure of the lasso-shaped molecules is shown in FIG. 1A and FIG. 4B.
Balloon-shaped closed oligonucleotides as described in FIG. 1A, FIG. 1B and FIG. 4C also form part of the invention. These oligonucleotides are closed with a chemical bond corresponding to an inter-chain bridging produced between two internal nucleotides. This bridging may be effected by means of a reactivexe2x80x94for example photoactivablexe2x80x94nucleotide, or alternatively by using an exogenous reagent which establishes a link between two paired regions of the oligonucleotide. Such a balloon-shaped oligonucleotide possesses only a limited number of substrate sites, or none, for exonucleases. Even if the bridging(s) closing the oligonucleotides molecule are not effected at the terminal nucleotide, only the few nucleotides localized on each side of the bridging point, at the ends of the molecule, will be accessible to the exonucleases. The exonucleases may cleave the phosphodiester bonds linking the nucleotides of the uncoupled ends, but will be stopped from the bridging site onwards. The nucleotides engaged in the bridging are totally or partially exonuclease-resistant, and thus protect the oligonucleotide against continued nucleolytic degradation.
Another family of circular oligonucleotides, closed up on themselves through a non-nucleotide molecule, also form part of the invention. These closed oligonucleotides possess a portion of their molecular structure corresponding to a DNA or RNA sequence in which the bases are unmodified and whose first nucleoside unit is linked to the last via a bond involving a molecular structure of any kind. For example, the antisense oligonucleotides may be circularized by means of a coupling between the terminal nucleotides via a protein or polypeptide structure which will be linked to the terminal nucleoside units via any type of coupling (FIG. 4). Circular oligonucleotides containing a nucleotide portion and a protein portion hence also form part of the invention. The insertion of this protein portion may be effected by various coupling methods. The protein fraction may be designed to increase the efficacy of the nucleotide molecule by different mechanisms. The protein portion may, for example, promote internalization of the oligonucleotide in cells, and enable certain cells to be targeted, where appropriate, by choosing a protein determinant used. The protein or peptide components used in the circular oligonucleotides of this type may also be signaling molecules, permitting an intracellular targeting of the oligonucleotides. For example, peptides targeted on the nucleus, originating from natural cellular or viral proteins, may be used. This aspect of the invention hence consists in proposing oligonucleotides of novel structure containing compounds capable of reacting specifically with particular receptors of the cell membrane surface, of being internalized by said cells and of exerting their biological activity inside the cell.
Circular oligonucleotides closed up on themselves via lipophilic groups or chains containing lipophilic radicals and promoting cellular internalization of these molecules also form part of the invention.
Closed oligonucleotides combined via covalent or non-covalent couplings with liposomal type encapsidation structures or any other lipoprotein or lipopolysaccharide structure also form part of the invention.
Circular oligonucleotides closed up on themselves via a phosphodiester bond, but additionally containing one or more internal bonds produced by reactive agents belonging to the structure of the molecules themselves or supplied exogenously, also form part of the invention.
These molecules will possess the characteristics of exonuclease resistance of circular oligonucleotides, and offer, in addition, particular secondary conformations capable of being adapted to the recognition of particular sites on targets themselves possessing particular secondary or tertiary conformations, whether these targets are nucleic acids or any other cellular or viral structure capable of possessing an elective and selective affinity with respect to a polynucleotide of particular primary and secondary structure.
Circular or closed antisense oligonucleotides capable of forming a triple helix with the target RNA also form part of the invention. The formation of a triple helix enables the DNA/RNA interaction between the antisense nucleotide and the target sequence to be stabilized.
Generally speaking, a circular antisense nucleotide may also be said to be a xe2x80x9cprodrugxe2x80x9d compound with respect to a linear antisense oligonucleotide. The cleavage of a circular oligonucleotide will give rise to a linear oligonucleotide which may exert a conventional antisense effect, with delay.
Although the compounds according to the invention will quite often contain nucleotide sequences incapable of self-pairing, closed oligonucleotides containing self-paired regions forming a double-stranded structure of variable length (see FIG. 5) also form part of the invention. This double-stranded structure may have several roles, for example that of stabilizing the molecule to permit its circularization during the synthesis of circular molecules (see the preparation methods later). This double-stranded structure may also have an active role, such as, for example, that of interacting with proteins possessing an affinity for this sequence. The double-stranded structure could correspond to a binding sequence for protein factors. In particular, an aspect of the invention is to provide stable, natural oligonucleotide molecules capable of binding cellular or viral protein factors and hence of interfering with biological processes involving these factors.
An example of the use of closed oligonucleotides containing a self-paired regionxe2x80x94oligonucleotides intended for such an application will advantageously be simply circularxe2x80x94is intracellular competition with transcription factors. In this case, the double-stranded portion of the closed oligonucleotide will carry the binding sequence of the transcription factor, regulator or hormonal nuclear receptor which it is desired to trap. Interaction between the circular oligonucleotide made double-stranded and a transcription factor will lead to a reduction in the intracellular availability of this factor, and will hence modify the regulatory equilibria in which this factor participates. If it is a positive transcription factor, exercising a stimulatory effect, blockade of this factor will lead to an inhibition of the genes in question; if it is a negative factor, inhibiting gene expression after interaction with the cellular DNA, expression of these genes will then be stimulated. Generally speaking, closed oligonucleotides of the circular type, of the deoxyribo-series and containing a double-stranded portion, may interact for therapeutic purposes with any protein factor possessing an affinity for DNA and whose effects in a given pathological situation it is desired to reduce or change.
The use is also envisaged of closed, advantageously circular oligonucleotides for combining antisense and sense approaches for the purpose of greater efficacy. This method consists in simultaneously using a closed antisense oligonucleotide directed towards the messenger RNA of a transcription factor, and a closed xe2x80x9csensexe2x80x9d oligonucleotide containing a double-stranded structure of sequence corresponding to the binding site of the transcription factor on the DNA. Two levels of action are hence targeted simultaneously and synergistically on the same target molecule, the antisense decreasing the synthesis of this factor and the sense molecule exerting a trapping effect on the residual molecules capable of persisting. The combined sense and antisense approaches may be used either to target the same protein at two different levels of expression and functionality, or to attack two different proteins for the purpose of seeking the greatest efficacy of the method.
This dual approach can make simultaneous use of two different molecules, one sense and the other antisense, or alternatively of a single closed oligonucleotide molecule like that described in FIG. 1C.
Another example of the use of closed oligonucleotides is intracellular competition with affinity factors for certain double-stranded RNA sequences. This is case with certain regulatory proteins which interact with messenger RNAs, especially with viral messenger RNAs. The case of the product of the HIV tat gene, which binds to a double-stranded region the the 5xe2x80x2 end of the viral messenger RNA, may be mentioned for example. To be used as agents interacting with tat, in the case of this example, the closedxe2x80x94for example circularxe2x80x94oligonucleotides would comprise a double-stranded portion of the ribo-series, the remainder of the oligonucleotide being composed of nucleotides either of the ribo-series or the deoxyribo-series.
Generally speaking, the closed oligonucleotides forming the subject of the invention may be used as stabilized molecules for trapping protein factors possessing an affinity for single-stranded or double-stranded nucleic acids, DNA or RNA, by mimicking both the primary structure (sequence) of the affinity sites and the possible secondary structures (hairpin structures, cruciform structures for example).
Such closed, advantageously circular, oligonucleotides, containing a nucleotide sequence recognized by protein factors, may also carry reactive groups capable of effecting bridgings, spontaneous or photo-activable, with the proteins interacting with them.
An aspect of the invention, as described in detail in the previous section, is hence to provide novel stable, natural oligonucleotides capable of being internalized in cells and of then interacting with cellular or viral factors possessing an affinity for specific nucleic acid sequences.
Circular oligonucleotides combined in pairs as a result of the complementarities of their sequences, to form a complete or partial double-stranded circular structure (capable of possessing non-pairings otherwise known as xe2x80x9cmismatchesxe2x80x9d) resistant to exonucleases, also form part of the invention. These circular double-stranded oligonucleotides may be composed of natural, rare, artificial or modified nucleotides of the deoxyribo-series or of the ribo-series. These double-stranded circular oligonucleotides could be composed of form I DNA or form V DNA, that is to say containing composite B- and Z-type structures. Form I DNA is super-coiled circular DNA whose two strands possess an interlacing. Form V DNA is a DNA whose complementary strands are not interlaced, that is to say a DNA formed from two complementary strands side by side. Double-stranded DNA can adopt different conformations, A, B or Z. The most natural and most common form of DNA is a B-type right-handed helix, whereas the less frequent Z form is a left-handed helix, more elongated than the B form. A circular structure such as that described above, containing two complementary strands not interlaced, will contain both left-handed helices and right-handed helices. Such a double-stranded circular oligonucleotides may be used as a sense type agent to interact with proteins possessing affinity for a particular sequence which will be present on the oligonucleotide in question. Such a circular double-stranded oligonucleotide, of the ribo-series in particular, may also be used generally as an immuno-stimulant, and more especially as an interferon-inducing agent. It should be noted that this potential immuno-stimulation function possessed by some RNAs may also be exploited using single-stranded circular oligonucleotides possessing self-paired double-stranded structures, as a result of the internal sequence complementarities and by turning to good account the advantage conferred by the exonuclease resistance of a circular structure.
The closed oligonucleotides which form the subject of the invention may be obtained chemically, biologically or by approaches making use of combinations of the techniques of synthetic chemistry and molecular biology.
The closed oligonucleotides may hence be prepared either from linear oligonucleotides, then closed up by chemical or biological techniques, or directly as oligo-nucleotides by chemical means, using reactions leading to cyclization of the molecule. These two approaches are considered successively below.
Various methods of chemical synthesis of natural oligonucleotides have been developed, and are well known to specialists working according to the rules of the art. For example, one method consists in using a solid support known as CPG (controlled pore glass), to which the first nucleotide is bound covalently by a coupling arm through its 3xe2x80x2-OH end. The 5xe2x80x2-OH end of the nucleoside is protected by an acid-labile di-p-methoxytrityl group. This approach, employing phosphite triester chemistry and in which deoxynucleoside 3xe2x80x2-phosphoramidites are used as synthons, is termed the phosphoramidite method (Caruthers, 1985). This approach is the one most widely used at present and has the advantage of being completely automatic. The synthesis of an oligonucleotide from a first unit bound to CPG begins with a step of deprotection, during which the trityl group is removed, a nucleoside unit activated on its 5xe2x80x2 group is then added, the unreactive products are blocked and a fresh cycle of deprotection/activation/coupling then begins again. Typically, the addition of a deoxynucleotide takes place according to the following four steps: i) deprotection and removal of a dimethoxytrityl protective group with acid, ii) condensation of the resulting product with a deoxynucleoside 3xe2x80x2-phosphoramidite, giving a deoxynucleoside phosphite triester, iii) acylation, that is to say blocking of the 5xe2x80x2-hydroxyl groups which have not reacted and iv) oxidation of the phosphite triester to phosphate triester. The repetition of such cycles leads to synthesis of oligonucleotides capable of extending to more than 200 units.
To quote a second example, another approach used for the synthesis of oligonucleotides is that of phosphonate chemistry (Froehler et al., 1986). This approach begins with the condensation of a deoxynucleoside 3xe2x80x2-H-phosphonate with a deoxynucleoside coupled to a silica glass support. Successive condensation cycles lead to the synthesis of oligonucleotide H-phosphonates. These oligonucleotides are oxidized in one step to give the phosphodiesterd.
Using one or other of these techniques, or any other sequential procedure permitting the chemical synthesis of polynucleotide chains of predetermined sequence, linear oligonucleotides which it is possible to circularize by biological means, using ligation enzymes, are obtained. To be able to use ligases to close the oligonucleotides up on themselves, the oligonucleotides must contain a 5xe2x80x2-P terminal group, irrespective of whether the phosphorylation of the 51 terminal has been performed chemically, or alternatively biologically using a kinase (preferably polynucleotide kinase) and ATP or any other phosphate donor.
Different procedures suitable for advantageously performing the circularization of linear oligonucleotides are described below.
1-1xe2x80x94A linear oligonucleotide may be circularized by establishing a partially double-stranded structure, paired to permit the functioning of a ligase, such as T4 ligase, by means of a second oligonucleotide shorter than the first and of sequence complementary to the two ends of the first oligonucleotide. In this case, illustrated in FIG. 6, the second, small oligonucleotide acts as an adaptor and makes it possible to place end to end the two terminal nucleotides of the oligonucleotide to be circularized. The action of T4 ligase, or of any other ligation enzyme capable of exerting its action on DNA, then permits circularization by forming a phosphodiester bond between these two nucleotides. This ligation can take place in solution, the two oligonucleotides being mixed in a medium permitting hybridization and ligation under suitable temperature and concentration conditions favorable to intra-chain circularization and unfavorable to inter-oligonucleotide ligations which can decrease the yield of the actual circularization reaction.
1-2xe2x80x94A variant of the method described in (1) consists in performing the circularization of the oligonucleotide, still by means of an adaptor, but using an oligonucleotide bound to a support which can be, for example, nitrocellulose or a derivative, a nylon membrane, a glass support, a polysaccharide structure or any other support enabling a nucleic acid fragment to be bound covalently or non-covalently and permitting subsequent hybridization between this fragment and an oligonucleotide, and which is compatible with the action of a ligase. This procedure hence consists in binding the adaptor oligonucleotide to a support, either by means of a covalent bond or via non-covalent links. It will be advantageous to use covalent bonds enabling a large number of hybridization/ligation cycles to be performed with the oligonucleotide to be circularized. The adaptor oligonucleotide may be bound to its support either at a terminal nucleotide, directly or by means of a coupling agent, or via a nucleotide located at an internal position and carrying a reactive group. A diagram showing the principle of such a method is illustrated in FIG. 6. The advantage of binding the adaptor oligonucleotide is twofold: on the one hand this binding performed under controlled physical conditions (number of molecules bound per unit area) enables the incidence of concatamer formation during the hybridization reaction following inter-oligonucleotide bonding to be reduced, and on the other hand it facilitates the production of ligation reactors, employing the adaptor oligonucleotides a large number of times for multiple circularization cycles.
1-3xe2x80x94The oligonucleotides may be circularized by taking advantage of secondary structures deliberately introduced by providing for sequences capable of folding back on themselves, forming partial double-stranded structures. For example, FIG. 6 illustrates the case of a xe2x80x9cdumbbell-shapedxe2x80x9d oligonucleotide, containing a linear portion forming a loop and containing the antisense type sequence, a double-stranded region 9 base pairs long and a closing sequence composed of T5. This structure is capable of accomplishing self-pairings, thereby giving a molecule capable of being utilized as a substrate by a ligation enzyme such as T4 ligase. In this case, the yield of the ligation depends, inter alia, on the stability of the pairing at the double-stranded structure. Several pairing sequences were subjected to comparative studies, and one of the sequences permitting an advantageous circularization yield (of the order of 75%) is shown in FIG. 6. Although it does not matter which of the nucleotides might be used to produce a closing loop of variable length, from 4 to 8 residues, and mainly either A or T, will preferably be used.
The double-stranded pairing region may contain either a sequence used only for the formation of the xe2x80x9cdumbbellxe2x80x9d structure, or a sequence corresponding in part to the target region and which can potentially be displaced by an intermolecular hybridization.
The experimental conditions permitting effective circularization of the oligonucleotide whose sequence is given in FIG. 6 are described in detail later in the experimental part (see xe2x80x9cProperties and Advantages of the Closed Oligonucleotidesxe2x80x9d). In the case of this sequence, the circularization yield is of the order of 75%.
This technique was used to prepare the circular oligonucleotides which are dealt with in the experimental part.
1-4xe2x80x94The circular oligonucleotides may also be formed by a double-stranded structure closed up on itself at each end via a short loop of joining nucleotides. These oligonucleotides may be used for xe2x80x9csensexe2x80x9d type approaches such as those described above. A typical example of a sense oligonucleotide is illustrated in FIG. 6. This oligonucleotide contains a paired sequence of 24 nucleotides and two joining loops formed by T5, In the example given here, this circular oligonucleotide contains a double-stranded structure corresponding to the sequence for recognition of the hepatocyte transcription factor HNF-1. Such an oligonucleotide may be circularized simply by taking advantage of the double-stranded secondary structure formed by the complementary sequences. The closing point (that is to say the ends) of the oligonucleotide will be chosen so as to permit the greatest efficacy of circularization by intramolecular refolding. This point may be centered or more or less distal relative to the mid-point of the central secondary structure. It should also be noted that such oligonucleotides may be synthesized, not on the basis of an intramolecular reaction, but by an intermolecular reaction using two linear oligonucleotides folded back on themselves, possessing a partial double-stranded structure and generating cohesive ends capable of pairing with one another (see the diagram in FIG. 6).
1-5xe2x80x94A technique consisting in preparing two complementary oligonucleotides, one long and the other short, the second hybridizing in the central portion of the first, may be used to cyclize oligonucleotides which are to contain a double-stranded central region. By the action of T4 ligase, it is possible to join the two ends of the long oligonucleotide to the ends of the paired oligonucleotide, even if there are no sequence homologies permitting the formation of a self-pairing at the 3xe2x80x2 and 5xe2x80x2 distal sequences. Such a mechanism leads to the formation of a closed, circular oligonucleotide structure containing a central double-stranded portion.
1-6xe2x80x94For the preparation of circular oligonucleotides of the ribo-series (oligoribonucleotides) from linear oligoribonucleotides, the techniques which can be used are of the same kind as those described above. However it is also possible to use a enzyme, T4 RNA ligase, which is capable of spontaneously accomplishing circularization of oligonucleotides of the ribo- or deoxyribo-series. This enzyme enables linear oligonucleotides to be closed and converted to circular oligonucleotides in the presence of ATP. It is hence possible, without a special matrix and in the absence of any adaptor oligonucleotide, to circularize oligoribonucleotides in this manner. This same enzyme also enables oligodeoxyribonucleotides to be circularide, to circula much lower efficacy than in the case of oligoribonucleotides. T4 RNA ligase will advantageously be employed to circularize antisense oligonucleotides possessing a ribozyme activity, or alternatively to circularize xe2x80x9csensexe2x80x9d RNAs such as, for example, the sequences interacting with HIV tat type transactivators.
1-7xe2x80x94The procedures described above all involve ligation enzymes to form phosphodiester bonds and to close the linear molecules. Any enzyme enabling a phosphodiester bond to be formed between two nucleotide residues may be used to circularize oligonucleotides for the purpose of making them resistant to nuclease action, in the spirit of the invention described here, be such enzymes DNA ligases or RNA ligases. In particular, heat-stable enzymes originating from heat-resistant organisms may advantageously be used, enabling successive ligation/denaturation/hybridization cycles to be performed. To prepare the circular molecules forming the subject of the invention, either ligation enzymes in solution, or enzymes bound to a support for the purpose of enhancing the yield of the reaction and/or decreasing its cost, may be used.
Any chemical reagent permitting chemical ligation may also be used to prepare the circular molecules that form the subject of the invention. For example, and without implied limitation, reagents such as carbodiimide or cyanogen bromide could be used.
The preparation of closed oligonucleotides for the purpose of pharmacological application, whether for animal experiments or for the preparation of pharmaceutical compounds, will advantageously employ chemical methods enabling the large quantities required to be prepared.
Several methods may be used: in particular, either linear oligonucleotides may be synthesized by the usual methods and then closed by chemical ligation or via a bond involving terminal nucleotides, or oligonucleotides capable of being subjected to a direct cyclization in the final step of the chemical synthesis may be synthesized.
Several procedures for preparing cyclic oligonucleotides have been described (Barbato et al., 1989, de Vroom et al., 1988). These approaches, in the liquid phase or on a support, enable short cyclic oligonucleotides to be obtained. For example, it is possible to use a technique which consists in binding a first nucleotide residue through the exocyclic amine group. From such a support, assembling of the oligonucleotide can take place from both 3xe2x80x2 and 5xe2x80x2 ends, which are protected and available. The cyclization is performed, when the synthesis is complete, using, for example, MSNT after unblocking the protective groups from the two ends.
To synthesize cyclic oligonucleotides for the purpose of manufacturing exonuclease-resistant antisense or sense molecules, any method of synthesis permitting the elongation of a polynucleotide chain from both 3xe2x80x2 and 5xe2x80x2 ends, and the joining of these two ends after completion of the elongation of the chosen sequence, may be used. Any method permitting the chemical joining of two independently synthesized linear oligonucleotides, subsequently recombined to form a closed structure by means of a specific chemical reaction of the ends, may be used.
Various methods may be used to close linear oligonucleotides in order to form closed structures forming the subject of the invention. In these structures, one or other or both of the two terminal nucleotides will be engaged in a coupling bond. Apart from the strictly circular structures described above, and which may be obtained either by ligation or by any other chemical reaction, several of the other closed structures already mentioned may be obtained by chemical coupling.
This is the case, in particular, with the lasso structures, or [sic] one of the terminal nucleotidesxe2x80x94advantageously the 3xe2x80x2 terminal nucleotidexe2x80x94is coupled to one of the nucleotides of the 5xe2x80x2 portion of the oligonucleotide. Such structures may be obtained using modified nucleotides capable of establishing bridgings with other portions of the molecule.
It is also the case with the balloon structures, in which a bridging has been established by any agent between two or more than two nucleotides located in the terminal regions of the oligonucleotide, these regions being paired [lacuna] a small number of nucleotides, preferably over 4 to 10 nucleotides.
It is also the case with the oligonucleotides which will be closed through any group, any molecule or macromolecule permitting a coupling of the terminal nucleotides with one another, or between a terminal nucleotide and an internal nucleotide, and capable of increasing the efficacy of the compound in terms of its antisense or sense intracellular action. As an example, polypeptides for example from 5 to 100 amino acids long may be used to cyclize the oligonucleotide, it being necessary for the polypeptide linkage to have a sufficient mass to be recognized by cell receptors and to permit a better internalization or a better targeting.
The closed, especially circular, oligonucleotides forming the subject of the invention described here may be used in all cases where it will be advantageous to have at one""s disposal an oligonucleotide possessing an increased resistance to exonucleases compared with a linear oligonucleotide.
The closed oligonucleotides may, in particular, be used as antisense or sense agents to act specifically on the transcription or translation of protein(s) whose level of expression it is desired to modulate for the purpose of research or therapy.
The few possible applications which are given below are merely non-exhaustive examples, in no way limiting, of situations in which it might be possible to envisage an antisense, or sense, type approach, and where the use of natural exonuclease-resistant oligonucleotides possessing the lowest possible toxicity would offer an advantage.
More especially, the compounds according to the invention are usable as a therapeutic agent, in particular as an antiviral or anticancer agent, especially in pharmaceutical compositions for external topical application or for systemic use.
However, they can also be inducers of natural immunomodulators such as interferon. It is also possible to use them in diagnosis in vitro or in vivo.
Generally speaking, the closed oligonucleotides, natural and circular in particular, will find especially suitable spheres of application in the field of dermatology, on account of the accessibility of the targets to be treated and the minimal or nonexistent toxicity of these compounds. All dermatological conditions which may be dependent on a mechanism of genetic dysfunction and for which an etiological factor may be identified and whose gene and/or messenger RNA sequence may be known will enable an antisense approachxe2x80x94or even a xe2x80x9csensexe2x80x9d approach in some favorable casesxe2x80x94to be potentially envisaged.
The use of natural oligonucleotides, which hence possess the lowest toxicity, enables the possibility of this kind of approach to be envisaged for serious or minor conditions, and even, where appropriate, for uses of the cosmetological type, that is to say on healthy skin and in a field of application where the toxicities of the products must be as low as possible.
Besides the viral targets defined later, many dermatological diseases could be treated with exonuclease-resistant circular or closed natural oligonucleotides. Among the potential targets of such approaches, inflammatory diseases such as atopical dermatitis or lupus erythematosus, keratinization diseases such as ichthyosis and psoriasis and neoplastic diseases such as melanoma or cutaneous T lymphoma. Thus, for example, circular antisense oligonucleotides applied in dermatology could be directed towards messenger RNAs of inflammation mediators such as interleukins, towards messenger RNAs of proteins involved in disorders of proliferation of the epidermal cells or alternatively towards messenger RNAs coding for proteins possibly involved in phenotypic skin aging, such as, for example, collagenase, elastase and transglutaminases.
More generally speaking, the closed oligonucleotides, natural and circular in the main, could, for example, be used as antisense, or sense, antiviral agents, either for topical (dermatological) indications or for systemic indications. For example, such oligonucleotides could be used as antiherpetic (HSV-1 and HSV-2, CMV, EBV) agents, as antipapillomavirus (cutaneous, genital, laryngeal or other HPV) agents, as antihepatitis (HBV, HCV, HDV) agents, as anti-HIV (HIV-1 and HIV-2) agents, as anti-HTLV (HTLV-1 or HTLV-2) agents, and the like.
These circular oligonucleotides could also be used as agents for repressing the expression of certain cellular proteins directly responsible for or involved in the etiology of diseases of cell proliferation and differentiation. For example, these circular oligonucleotides could be directed towards the expression of cellular oncogenes which are hyperexpressed or expressed in an uncontrolled manner in tumor cell types (RAS, ERB, NEU, SIS, MYC, MYB, and the like).
In particular, these natural circular oligonucleotides resistant to serum exonucleases could be used as antisense agents directed towards messenger RNAs of oncogenes expressed in leukemic cells and involved in their proliferation, or alternatively as xe2x80x9csensexe2x80x9d agents directed towards proteins having affinity for certain DNA sequences and expressed at pathological levels in some of these proliferative diseases. In the context of the treatment of certain leukemias, for marrow transplants, the circular, closed oligonucleotides may be used in the context of xe2x80x9cex vivoxe2x80x9d applications.
For these many indications, it will be possible to establish appropriate pharmaceutical dosage formulations in order to optimize the delivery of these molecules to their target cells. Thus, for example, closed, especially circular, oligonucleotides may be encapsulated in liposomes, nanoparticles or LDL particles, or in any other type of microsphere permitting appropriate preservation and promoting targeting. The closed, for example circular, oligonucleotide molecules could also be combined with cationic surfactants. It is quite obvious that these few examples are neither exhaustive nor limiting.
The closed, especially circular, oligonucleotides forming the subject of the invention described here are hence capable of being included in all kinds of pharmaceutical preparations, at concentrations which vary according to the indication.
In particular, the dermatological applications mentioned above will require the preparation of creams, solutions, emulsions, lotions, gels, sprays, powders, aerosols, and the like, prepared by combining the circularxe2x80x94or closedxe2x80x94oligonucleotide of chosen sequence with the common pharmaceutical components participating in the composition of these products. For example, for preparations intended for topical applications in dermatology, the circular, or closed, oligonucleotides may be combined with all kinds of preservatives such as methyl or propyl hydroxybenzoate or benzalkonium chloride, for example, and other stabilizing, emulsifying, dispersing, suspending, solubilizing, coloring and thickening agent, fragrances, and the like. It should be noted that some of these compositions, especially the compositions intended for topical applications for dermatological indications, may combine both circularxe2x80x94or closedxe2x80x94oligonucleotides with other active principles such as, for example, bacteriostatic or antiseptic or antimicrobial or antibiotic or analgesic or antipruritic agents, and the like.
All these examples are given only to illustrate the intention and are neither exhaustive nor limiting.
The experimental examples are given below to illustrate the advantages of a closed oligonucleotide over a linear xe2x80x9copenxe2x80x9d oligonucleotide of the same sequence. These examples are taken from experiments carried out with oligonucleotides circularized according to the method described in the part xe2x80x9cproduction of closed oligonucleotidesxe2x80x9d, section 1-3. The closed oligonucleotides dealt with here are hence circular oligonucleotides composed of natural nucleotides linked to one another via unmodified phosphodiester bonds.